Home › Cell Biology › Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells
Cell Biology JoVE (Open Access) Citable · DOI

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

DOI: 10.3791/50881-v
What you'll learn
  • ✓ Establish organotypic brain slice cocultures with carcinoma cell spheroids
  • ✓ Visualize carcinoma cell invasion dynamics using fluorescence and bright-field microscopy
  • ✓ Perform immunofluorescence staining and imaging of brain resident immune cells
  • ✓ Analyze morphological changes and cell-cell interactions in brain-tumor coculture systems
Protocol

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Difficulty
advanced
Total time
~5–7 days per coculture experiment (slice preparation ~1 day, coculturing ~3–5 days, immunostaining and imaging ~1–2 days)
Model organism
Mouse (organotypic brain slices); carcinoma cell spheroids (human or murine)
Biosafety
BSL-2

Steps

1
Prepare and establish organotypic brain slice cultures

Isolate mouse brain tissue and culture as organotypic slices on membrane inserts. Maintain slices in appropriate culture medium for 3–5 days prior to coculturing.

▶ 01:41
2
Coculture organotypic slices with carcinoma cell spheroids

Place 3D carcinoma spheroids onto organotypic brain slices and monitor invasion and morphological changes over the coculture period using video and fluorescence microscopy.

▶ 04:11
3
Perform immunofluorescence staining and confocal imaging

Fix cocultures and stain for astrocytes, microglia, and other cellular markers. Image using fluorescence microscopy to visualize cell interactions and invasion dynamics.

▶ 05:42
4
Analyze and interpret coculture morphology results

Document representative images and datasets demonstrating carcinoma invasion patterns, astrocyte and microglial responses, and quantify morphological changes in the coculture system.

▶ 07:11
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